Nanowire-based system for analysis of nucleic acids

ABSTRACT

System for detection and/or analysis of nucleic acids using nanowires to detect covalent modification of nucleic acids.

CROSS-REFERENCE TO PRIORITY APPLICATION

This application is a continuation of U.S. application Ser. No.11/394,147, filed Mar. 29, 2006, and claims the benefit under 35 U.S.C.§119(e) of U.S. Provisional Patent Application Ser. No. 60/666,396,filed Mar. 29, 2005, which is incorporated herein by reference in itentirety for all purposes.

INTRODUCTION

Characterization of nucleic acid sequences has widespread application ina growing number of areas, including clinical diagnostics andtherapeutics, forensics, and analysis of bio-terrorism agents, amongothers. For example, the relatively new field of pharmacogenetics isbased on the recognition of a strong genetic component to theeffectiveness of medical treatments. In particular, nucleic acidsequence differences between members of the human population can providemuch of the variation in response of the population to a medicaltreatment, such as a drug. Nucleic acid sequence analysis of prospectivedrug recipients thus can be used to pair each recipient moreintelligently with a drug based on the recipient's genetic makeup.However, current sequencing technologies may be limited in their abilityto meet the growing demand for sequence information driven bypharmacogenetics and numerous other applications.

SUMMARY

The present teachings provide a system for detection and/or analysis ofnucleic acids using nanowires to detect covalent modification of nucleicacids.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic view of an exemplary system for nanowire-baseddetection and/or analysis of nucleic acids, in accordance with aspectsof the present teachings.

FIG. 2 is a flowchart of an exemplary method of nanowire-based analysisof nucleic acids, in accordance with aspects of the present teachings.

FIG. 3 is a somewhat schematic view of an exemplary array of nanowireassemblies that can be included in the systems of the present teachings.

FIG. 4 is a somewhat schematic view of an exemplary approach forcoupling templates to nanowires to create an array of nanowireassemblies, in accordance with aspects of the present teachings.

FIG. 5 is a somewhat schematic view of an exemplary approach forcoupling primers to nanowires to create an array of nanowire assemblies,in accordance with aspects of the present teachings.

FIG. 6 is a somewhat schematic view of an exemplary system fornanowire-based sequencing of nucleic acids, in accordance with aspectsof the present teachings.

FIG. 7 is a flowchart of an exemplary method of nanowire-basedsequencing of nucleic acids, in accordance with aspects of the presentteachings.

FIG. 8 is a somewhat schematic view of an exemplary approach tosequencing nucleic acids using an array of nanowire assemblies and of agraph of exemplary data that may be obtained with this approach, inaccordance with aspects of the present teachings.

FIG. 9 is a somewhat schematic flowchart of an exemplary method ofnanowire-based analysis of nucleic acids using ligation, in accordancewith aspects of the present teachings.

FIG. 10 is a somewhat schematic flowchart of an exemplary method ofnanowire-based analysis of nucleic acids using cleavage, in accordancewith aspects of the present teachings.

FIG. 11 is a somewhat schematic view of an exemplary nanowire assemblyduring coupling of a nucleic acid (an analyte and/or a probe) to ananowire of the assembly at two or more spaced sites along the nucleicacid, in accordance with aspects of the present teachings.

DESCRIPTION OF VARIOUS EMBODIMENTS

The present teachings provide a system for detection and/or analysis ofnucleic acids using nanowires to detect covalent modification of nucleicacids. Each nanowire can be coupled to a nucleic acid analyte and anucleic acid probe base-paired with the analyte, to form a nanowireassembly. The nanowire assembly or any array of nanowire assemblies canbe contacted with a reagent for covalent modification of nucleic acidsbased on nucleic acid structure. For example, the reagent can include anucleic-acid modifying enzyme, such as a polymerase, a ligase, or anuclease, among others, capable of lengthening or shortening nucleicacids. A change in the size of a nucleic acid(s) coupled to a nanowireassembly can change the electrical characteristics, such as theconductance, of the nanowire assembly Accordingly, a detector can beused to measure an electrical characteristic of the nanowire assembly,to determine whether or not the electrical characteristic has beenchanged by action of the reagent. The presence or absence of change inthe electrical characteristic (and/or the size (and/or polarity) of anychange) thus can provide structural information, such as sequenceinformation, about the analyte.

Overall, nanowire-based analysis of nucleic acids can have a number ofadvantages over other systems. These advantages can include increasedsensitivity, analysis of smaller amounts of analytes, multiplexedanalysis of analytes, performance of a large number of analyses (e.g.,hundreds or thousands) in a small space, decreased size ofinstrumentation, improved portability, and/or the like. For example, incontrast to chain termination approaches to sequencing (e.g.,dideoxynucleotide-based sequencing), the nanowire-based systemsdescribed herein can sequence nucleic acids by successive primerextension and measurement. Accordingly, the same individual primer andtemplate molecules can be involved in determining the identity ofdifferent nucleotides in the template, thereby substantially reducingthe number of primer and template molecules necessary for sequencing.

FIG. 1 shows an exemplary system 20 for nanowire-based analysis ofnucleic acids. The system can include a reagent delivery system 22, ananowire assembly 24, a detector 26, and/or a controller 28.

Reagent delivery system 22 can transfer one or more reagents 30 fornucleic acid modification, and particularly fluid reagents, to and/orfrom the nanowire assembly, shown at 23. The reagent delivery system canbe a flow-based system including a pump(s), a valve(s), one or morereservoirs, a channel(s) in which the nanowire assembly is disposed,and/or the like. Further aspects of the reagent delivery system aredescribed, for example, in Section V and in Example 3.

Nanowire assembly 24 can provide a site for nucleic acid modification.In particular, the nanowire assembly can include a nanowire 32 and oneor more nucleic acids 34 coupled to the nanowire, shown at 36. Thenucleic acids can include an analyte 38, which is the subject of theanalysis, and a probe 40, which facilitates structural analysis of theanalyte. The probe can be configured to form base pairs with theanalyte, shown at 42, so that the probe can couple the analyte to thenanowire, as in the present illustration, or vice versa, among others.In some embodiments, the system can include a plurality of nanowireassemblies, to form an array of nanowires (and nanowire assemblies).Further aspects of nanowires, nanowire arrays, nucleic acids, andcoupling nucleic acids to nanowires are described, for example, inSections I and II and in Examples 1 and 2, among others.

Detector 26 can measure a characteristic such as an electricalcharacteristic of the nanowire assembly. Accordingly, the detector canbe coupled electrically to the nanowire, shown at 44, for example,through a pair of electrodes disposed at spaced positions along thenanowire. In some examples, the detector can measure electricalcharacteristics from each of a plurality of nanowires disposed in anarray, for multiplexed analysis of nucleic acids. Further aspects ofdetectors are described, for example, in Section IV.

Controller 28 can control various aspects of system operation. Forexample, the controller can be coupled to the detector, shown at 46, todetermine when the detector measures the electrical characteristicand/or on which nanowire assembly of an array. The controller also canstore and/or process data received from the detector, such as datacorresponding to measured electrical characteristics. The controlleralso, or alternatively, can be coupled to the reagent delivery system,shown at 48, to control and/or monitor delivery of reagents to/from thenanowire assembly. Further aspects of controllers are described, forexample, in Section VI.

FIG. 2 is a flowchart 60 illustrating an exemplary method ofnanowire-based analysis of nucleic acids. The method can include stepsof (1) providing a nanowire assembly, shown at 62, (2) contacting withnanowire assembly with at least one reagent, shown at 64, and (3)measuring an electrical characteristic of the nanowire assembly, shownat 66. These steps can be performed in any suitable order, in anysuitable combination, and any suitable number of times.

A nanowire assembly can be provided. The nanowire assembly can include anucleic acid analyte coupled to a nanowire, either directly and/orthrough a nucleic acid probe that pairs (hybridizes) with the analyte.The step of providing can include or be preceded by a step of forming ananowire assembly. The step of forming a nanowire assembly can includecoupling nucleic acids covalently and/or noncovalently to a nanowire. Insome examples, an array of nanowire assemblies can be provided. Furtheraspects of providing and forming a nanowire assembly are described, forexample, in Sections I-III and in Examples 1 and 2.

The nanowire assembly can be contacted with a reagent for covalentmodification of nucleic acids based on nucleic acid structure. Forexample, the reagent can include an enzyme (such as a polymerase,ligase, nuclease, etc.) and/or a nucleotide monomer or polymer, amongothers. A covalent modification can include nucleotide addition to, orremoval from, the analyte and/or probe. Accordingly, the structure ofthe analyte can determine, for example, presence or absence of thecovalent modification, the position of the covalent modification (suchas position within the analyte and/or probe), and/or the extent of thecovalent modification (such as number of nucleotides added or removed).Further aspects of reagents and covalent modification of nucleic acidsare described, for example, in Section V and in Examples 3-5, amongothers.

An electrical characteristic of the nanowire assembly can be measured.The electrical characteristic, and particularly a change in theelectrical characteristic, if any, can be used to determine whether ornot the analyte has a structure that allows covalent modification by thereagent, thus providing information about the analyte's structure.Further aspects of measuring electrical characteristics and determininganalyte structure based on changes in an electrical characteristic aredescribed, for example, in Section IV and in Examples, 3-5, amongothers.

The steps of contacting and measuring can be repeated any suitablenumber of times. In some examples, contacting and measuring can berepeated with different reagents (such as different enzymes and/ornucleotide substrates) and/or can be repeated cyclically with the samesubstrate multiple times, such as for determining the sequence of ananalyte region of two or more nucleotides.

The methods of the present teachings also or alternatively can include astep of adjusting the stringency of base-pair interactions. Thestringency can be adjusted to alter the stability of nucleic acidstrand-strand interactions, such as to increase or decrease the totalnumber, uninterrupted length, and/or type of base-pair interactions(e.g., G-C base pairs are generally more stable than A-T base pairs)necessary to hold together nucleic acid strands that are complementary.In some examples, adjusting the stringency can include disruptingbase-pair interactions of nucleic acid duplexes, such that only lessstable duplexes and/or at least substantially all double-strandedduplexes are disrupted to form unpaired single strands. Adjustingstringency can be performed electrically (e.g., by positively ornegatively biasing a nanowire assembly), by changing the temperature ofthe nanowire assembly (e.g., by heating or cooling a correspondingreaction compartment and/or fluid disposed in, or destined for, thecompartment), and/or chemically (e.g., by adjusting ionic strength, theconcentration of divalent or multivalent cations and/or anions, thesolvent dielectric constant (e.g., by changing the concentration ofdimethylformamide or another organic solvent), and/or enzymatically(e.g., by adding or adjusting the concentration of enzymes such ashelicase that favor the pairing or unpairing of nucleic acid bases orstrands), and/or changing the concentration of chaotropic agents (suchas urea), among others). Nucleic acid duplexes can be disrupted, forexample, after contacting and before measuring (e.g., to remove acontribution to the electrical characteristic produced by nucleic acidduplexes), or after contacting and after measuring (e.g., to facilitateperformance of another cycle of contacting and measuring). In someexamples, adjusting the stringency can include reducing the ionicstrength of fluid in contact with the nanowire assembly to increase thesensitivity of measuring the electrical characteristic.

Further aspects of the present teachings are described in the followingsections, including (I) nanowires; (II) nucleic acids, including (A)analytes and (B) probes; (III) nanowire assemblies; (IV) detectors; (V)reagent delivery systems; (VI) controllers; and (VII) examples.

I. Nanowires

The systems of the present teachings include one or more nanowires. Ananowire, as used herein, is an elongate semiconductor having asub-micrometer cross-sectional dimension at one or more (or all)positions along its length. The cross-sectional dimension (and/ororthogonal cross-sectional dimensions) can be less than about 500 nm,100 nm, 20 nm, 5 nm, or 1 nm, among others.

The nanowires can have any suitable length. Exemplary lengths include atleast about 1 μm, 5 μm, or 20 μm, among others. Furthermore, thenanowires can have any suitable aspect ratio (length relative to across-sectional dimension (and/or relative to orthogonal cross-sectionaldimensions) to produce an elongate structure. Exemplary aspect ratiosinclude at least about 2:1, 10:1, 100:1, or 1000:1.

The nanowires can have any suitable cross-sectional shape. Exemplarycross-sectional shapes include circular, elliptical, polygonal(triangular, rectangular, etc.), irregular, and/or a combinationthereof. In some examples the nanowires can be nanotubes having a hollowcore.

The nanowires can be formed of any suitable material(s). For example,the nanowires can be formed of semiconductor materials (elements oralloys), with or without a dopant. Exemplary semiconductor materials toform the body (or a coating or lining) of the nanowire include silicon,germanium, and/or carbon, among others. Exemplary dopants include n-typedopants and/or p-type dopants, such as nitrogen and phosphorus,respectively, among others. Other materials that can be suitable to formthe body (and/or coating or lining) of the nanowires or as dopantstherein are described in the following patent applications, which areincorporated herein by reference: Serial No. 09/935,776, filed Aug. 22,2001 (Pub. No. US 2002/0130311 A1); Ser. No. 10/020,004, filed Dec. 11,2001 (Pub. No. US 2002/0117659 A1); Ser. No. 10/033,369, filed Oct. 24,2001 (Pub. No. US 2002/0130353); and Ser. No. 10/196,337, filed Jul. 16,2002 (Pub. No. US 2003/0089899 A1).

II. Nucleic Acids

The systems of the present teachings provide nanowires coupled tonucleic acids. A nucleic acid (or an oligonucleotide, an oligomer, or apolynucleotide), as used herein, is a polymer of at least two nucleotidesubunits linked together. The nucleic acid can be single-stranded ordouble-stranded (a duplex), among others. Double-stranded nucleic acidsgenerally are formed by hydrogen-bonding (base-pairing) between alignednucleotides of paired strands of nucleic acids, for example, adenosine(A) paired with thymidine (T) (or uridine (U) in RNA), and guanosine (G)paired with cytidine (C), among others.

The nucleic acid can have any suitable natural and/or artificialstructure. The nucleic acid can include a sugar-phosphate backbone ofalternating sugar and phosphate moieties, with a nucleotide baseattached to each sugar moiety. Any sugar(s) can be included in thebackbone including ribose (for RNA), deoxyribose (for DNA), arabinose,hexose, 2′-fluororibose, and/or a structural analog of a sugar, amongothers. The nucleotide base can include, for example, adenine, cytosine,guanine, thymine, uracil, inosine, 2-amino adenine, 2-thiothymine,3-methyl adenine, C5-bromouracil, C5-fluorouracil, C5-iodouracil,C5-methyl cytosine, 7-deazaadeine, 7-deazaguanine, 8-oxoadenine,8-oxoguanine, 2-thiocytosine, or the like. The nucleic acids of thepresent teachings can include any other suitable alternative backbone.Exemplary alternative backbones include phosphoramides,phosphorothiozates, phosphorodithioates, O-methylphosphoroamidites,peptide nucleic acids, positively charged backbones, non-ribosebackbones, etc. Nucleic acids with artificial backbones and/or moietiescan be suitable, for example, to increase or reduce the total charge,increase or reduce base-pairing stability, increase or reduce chemicalstability, to alter the ability to be acted on by a reagent, and/or thelike.

A nanowire can be coupled to a nucleic acid analyte (or a plurality ofstructurally different analytes) and/or to a nucleic acid probe (or aplurality of structurally different probes). Furthermore, the nanowirecan be coupled to a single molecule or to a plurality of molecules ofeach analyte and/or probe.

A. Analytes

An analyte, as used herein, is a nucleic acid that is the subject of ananowire-based analysis. The analyte can be from any suitable source,can have any suitable structure, and can be analyzed for any suitablefeature. In some examples, the analyte can be a template, that is, anucleic acid used as a model or guide for forming at least a region ofanother nucleic acid. The template can, for example, direct addition ofone or more nucleotides to a probe, serially or in parallel, accordingto a complementary region of the template that base-pairs with the oneor more nucleotides.

The analyte can be from any suitable source. Exemplary sources caninclude a human subject, a nonhuman animal, a plant, a microorganism, aresearch sample, an environmental sample (such as soil, air, water,etc.), and/or in vitro synthesis, among others.

The human subject can be a disease patient, a genetic screening subject,a person to be identified, a forensic subject, and/or the like. Theanalyte can be obtained from any suitable site in the human subject,including a sample from blood, plasma, serum, sperm, urine, sweat,tears, sputum, mucus, milk, a tissue sample, a tumor biopsy, culturedcells, and/or the like.

The analyte can be obtained in any suitable form by any suitableprocessing. For example, the analyte can be included in a crude lysateor can be a purified analyte obtained, for example, by ion exchangechromatography, selective precipitation, centrifugation, and/oramplification (such as with the polymerase chain reaction), amongothers. The analyte can be single- or double-stranded and can have anysuitable size. In some examples, the analyte can have a single size or aset of sizes produced by shearing, restriction endonuclease digestion,in vitro synthesis, amplification, limited chemical digestion, and/orthe like. In some examples, the analyte coupled to a nanowire includes aplurality of discrete strands of similar or identical length andsequence content, or of distinct lengths and/or sequence content.Strands of distinct length can be overlapping fragments, for example,including a common region of similar or identical sequence, such as forhybridization (base-pairing) with a probe. The analyte can be anysuitable size relative to the probe. In some examples, the analyte isabout the same size as the probe. In some examples, the analyte islonger than the probe and can be substantially longer than the probe,such as at least about two, ten, or one hundred times as long.

The analyte can be analyzed to obtain structural information about anysuitable feature(s). Generally, the structural information relates to asequence feature. The sequence feature can be defined by any suitablelength of nucleotides. The structural information thus can be thepresence or absence of a sequence feature of interest, the nucleotideidentity (e.g., G, A, T, or C) at a particular position(s) within theanalyte (e.g., to characterize a single nucleotide corresponding to asingle nucleotide polymorphism in the population), and/or the particularsequence of a stretch of at least about 5, 10, 50, 200, or 1000nucleotides, among others, of the analyte. The sequence feature thus canbe compared to a known sequence, to look for nucleotide identity and/ordifferences, or can correspond to a previously unsequenced region of agenome or other polynucleotide structure.

B. Probes

A probe, as used herein, is a nucleic acid that facilitates analysis ofthe nucleic acid analyte. The probe can be from any suitable source, canhave any suitable structure, and can be used to analyze the analyte forany suitable feature(s).

The probe can be obtained from a natural and/or artificial source.Accordingly, the probe can be synthesized or formed by a cell(s), a celllysate(s), a synthetic enzyme(s), chemical synthesis, enzymaticcleavage, chemical cleavage, and/or ligation, among others. The probethus can be RNA, DNA, or any suitable artificial derivative thereof.Furthermore, the probe can belong to the same structural class ofmolecules as the analyte (e.g., each being DNA or each being RNA) or toa different class of molecules (e.g., the probe being DNA and theanalyte RNA (or vice versa), or the probe having an uncharged orpositively charged backbone and the analyte having a phosphodiesterbackbone, among others).

The probe can have any suitable structure relative to the analyte. Theprobe can be configured to form a duplex with the analyte throughbase-pair interactions, so that the probe and analyte together form anat least partially double-stranded nucleic acid. Accordingly, a section(or all) of the probe can be complementary to a section (or all) of theanalyte. Alternatively, or in addition, the probe can include adouble-stranded region, independent of the analyte, for example, tocouple the probe to a nanowire. The probe can be configured to hybridize(base-pair) to any region of the analyte, for example, the probe canhybridize adjacent an end or spaced from the end of the analyte. In someexamples, the probe can be a primer. The primer can be configured sothat the 3′-end of the probe is base-paired with the analyte and spacedfrom the ends of the analyte, allowing the 3′-end to be extended with apolymerase (or ligase) and a suitable nucleotide substrate(s). The probecan have any suitable length sufficient to form a duplex structure withanother nucleic acid, particularly the analyte. The duplex structure canbe stabilized, for example, by nucleotide addition to the probe (such aswith a polymerase or ligase, among others).

The systems of the present teachings can include one probe or aplurality of probes. The plurality of probes can be configured to formduplexes with different analytes, different regions of the same analyte,or with the same region of the same analyte (e.g., see Example 4).

III. Nanowire Assemblies

The systems of the present teachings can include one or more nanowireassemblies. The nanowire assemblies can be disposed in any suitablearrangement, can include any suitable number and type of nucleic acids,and can couple the nucleic acids to nanowires by any suitablemechanism(s).

The systems of the present teachings can include an array of nanowireassemblies. The nanowire assemblies can be arrayed in a lineararrangement, a two-dimensional arrangement, and/or a three-dimensionalarrangement (such as stacked two-dimensional arrays). The array caninclude any suitable number of assemblies, including at least about ten,one-hundred, or one-thousand, among others. The nanowire assemblies caninclude a different probe (or probes) or the same probe (or probes) ineach assembly, and/or a different analyte (or analytes) or the sameanalyte(s) or analyte region(s) in each assembly. Accordingly, thenanowire assemblies can be configured to analyze distinct regions(nonoverlapping or overlapping) of the same analyte, the same region ofthe same analyte, and/or different regions of different analytes.

The analyte and probe can be coupled to each nanowire by any suitablemechanism. The analyte and/or the probe can be coupled directly to thenanowire by a covalent or noncovalent mechanism. Covalent mechanismsinclude bond formation between any suitable reactive pair with pairmembers disposed on the nanowire and a nucleic acid. An exemplarymechanism includes reaction of1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) withnucleic acids to form nucleic acid derivatives that can react withactivated nanowire surfaces. Alternatively, or in addition, nucleicacids can be bound to nanowires through noncovalent specific bindingpair interactions. For example, a first member of a specific bindingpair can be attached to a nanowire and a second member of the specificbinding pair can be attached (or included in) a nucleic acid analyteand/or probe. Specific binding pairs generally undergo specific binding,that is, binding to one another to the exclusion of binding to mostother moieties. Specific binding can be characterized by a dissociationconstant or coefficient (alternatively termed an affinity or bindingconstant or coefficient). Generally, dissociation constants for specificbinding range from 10⁻⁴ M to 10⁻¹² M and lower, and preferreddissociation constants for specific binding range from 10⁻⁸ or 10⁻⁹ M to10⁻¹² M and lower. Exemplary specific binding pairs are presented inTable 1:

TABLE 1 Exemplary Specific Binding Pairs Specific Binding Member Partnercell-surface receptor secreted hormone or cell-associated ligand nuclearreceptor nuclear hormone or DNA antibody antigen avidin or streptavidinbiotin lectin or carbohydrate receptor carbohydrate DNA antisense DNA;protein RNA antisense or other RNA; protein enzyme enzyme substrate orregulator histidine NTA (nitrilotriacetic acid) IgG protein A or proteinGSpecific binding pair interactions (e.g., base pairing) also can be usedto associate the analyte and probe with each other. Accordingly, theprobe (or analyte) can be coupled more directly to the nanowire than theanalyte (or probe) with which it is base paired. Moreover, the probe andanalyte can be coupled to a nanowire at the same time (e.g., in abase-paired condition) or sequentially, for example, by coupling theprobe first and then the analyte, or vice versa (see Example 2).Furthermore, the probe and/or analyte can be coupled to the nanowire ata single site or at multiple sites (e.g., spaced sites) along eachmolecule of the probe/analyte (see Example 6).

Probes and/or analytes can contact nanowires to form nanowire assembliesat any suitable time using any suitable contact mechanism. In someexamples, the nanowires can be disposed in an array and then differentprobes and/or analytes selectively contacted with (and coupled to)individual nanowires or subsets of nanowires in the array. For example,individual probes and/or analytes (or different sets thereof) can beselectively dispensed to regions of the array in small droplets of fluidso that the regions remain in fluid isolation, such as by inkjetprinting technology (e.g., using a dispensing head with thin-film heaterelements and/or piezoelectric elements, among others, as used in inkjetprintheads). Alternatively, or in addition, the array of nanowires canbe contacted with the probes and/or analytes with the nanowires disposedin fluid communication. For example, the probes and/or analytes canselectively interact with the nanowires based on specific bindingpartners (and particularly nucleic acids) previously coupled to thenanowires.

Further aspects of nanowires, nanowire assemblies, and assays that maybe performed with nanowires are described in the following patentapplications, which are incorporated herein by reference: U.S.Provisional Patent Application Ser. No. 60/612,315; U.S. patentapplication Ser. No. 09/935,776, filed Aug. 22, 2001 (Pub. No. US2002/0130311 A1); U.S. patent application Ser. No. 10/020,004, filedDec. 11, 2001 (Pub. No. US 2002/0117659 A1); U.S. patent applicationSer. No. 10/033,369, filed Oct. 24, 2001 (Pub. No. US 2002/0130353); andU.S. patent application Ser. No. 10/196,337, filed Jul. 16, 2002 (Pub.No. US 2003/0089899 A1).

IV. Detectors

The systems of the present teachings generally include one or moredetectors (also termed sensors) to measure an electrical characteristicof each nanowire (and nanowire assembly) of a nanowire array. In someexamples, the detector is coupled electrically to each nanowire in aserial fashion, using, for example, electronic switching devices.Accordingly, the detector can be coupled to the nanowires in arepeatable cycle, and the electrical characteristic of each nanowire canbe detected periodically to measure any time-dependent (and generallyreagent-dependent) changes (if any) in the electrical characteristic.

The detector can measure any suitable electrical characteristic.Exemplary electrical characteristics include conductance, resistance,current, voltage, and/or the like. The electrical characteristic can bemeasured qualitatively (e.g., change or no change and/or a positive ornegative change) or quantitatively (e.g., to determine a magnitude ofthe change, if any). The electrical characteristic can provide an analogand/or digital output.

V. Reagent Delivery Systems

The systems of the present teachings can include one or more reagentdelivery systems. The reagent delivery systems can include one or morepumps, valves, fluid reservoirs, channels, and/or reagents, amongothers.

Pumps generally include any mechanism for moving fluid and/or reagentsdisposed in fluid. In some examples, the pump can be configured to movefluid and/or reagents through passages with small volumes (i.e.,microfluidic structures). The pump can operate mechanically by exertinga positive or negative pressure on fluid and/or on a structure carryingfluid, electrically by appropriate application of an electric field(s),or both, among others. Exemplary mechanical pumps may include syringepumps, peristaltic pumps, rotary pumps, pressurized gas, pipettors, etc.The mechanical pumps may be micromachined, molded, etc. An exemplaryperistaltic pump created with a fluidic layer and a control layer thatare elastomeric is described, for example, in U.S. Pat. No. 6,408,878,issued Jun. 25, 2002, which is incorporated herein by reference.Exemplary electrical pumps can include electrodes and may operate byelectrophoresis, electroendoosmosis, electrocapillarity,dielectrophoresis (including traveling wave forms thereof), and/or thelike.

Valves generally include any mechanism for regulating the passage offluid through a channel. The valves can include, for example, deformablemembers that can be selectively deformed to partially or completelyclose a channel, a movable projection that can be selectively extendedinto the channel to partially or completely block the channel, anelectrocapillary structure, and/or the like. The valves can be operable,for example, to select a reagent to be contacted with a nanowireassembly (or assemblies), from a set of available reagents. Accordingly,the valves can be operable to provide selective fluid communicationbetween a nanowire assembly (or assemblies) in a channel (a reactioncompartment) and two or more reagent reservoirs.

Fluid reservoirs generally include any compartments for holding reagentsbefore and/or after they have passed through the reaction compartmentholding one or more nanowire assemblies. The fluid reservoirs can haveany suitable volume. In some examples, the fluid reservoirs have avolume that is substantially larger than the volume of the reactioncompartment, such as a volume that is at least about ten-fold,one-hundred-fold, or one-thousand fold the reaction compartment volume.The fluid reservoirs can be configured to be accessible from outside thesystem, to facilitate adding or removing fluid, such as with a pipette.

Channels generally include any passages that allow flow of fluid and/ormovement of reagents. The channels can extend, for example, betweenfluid reservoirs of the system, and can define a reaction compartmentthat holds the nanowire assembly (or assemblies). The channels can haveany suitable dimensions. In some embodiments, the channels can bemicrofluidic channels. Microfluidic channels or compartments, as usedherein, can have a cross-sectional dimension, at one or more positionsalong their length, of less than about one micrometer or less than about100 nanometers.

Reagents can have any suitable function in nucleic acid analysis. Thereagents can be configured, for example, to structurally modify nucleicacids of nanowire assemblies, to wash out (remove) a previouslydispensed reagent and/or a released nucleic acid, to adjust thestringency of hybridization between paired nucleic acid strands, todisrupt base-pair interactions (denature duplexes into single strands)substantially or completely, and/or the like.

Reagents include any chemical substances that can contact nanowireassemblies to facilitate analysis of nucleic acids. The chemicalsubstances can be present in any suitable form in a reagent, includingas a mixture, a complex, a solution, a suspension, and/or the like.Exemplary reagents can include catalysts and/or mono- and/orpolynucleotides. Other exemplary reagent components can include carrierfluids (such as water and/or an organic fluid), enzyme cofactors (suchas divalent cations (e.g., magnesium, zinc, manganese, etc.),ribonucleoside triphosphates (such as adenosine triphosphate (ATP)),S-adenosyl methionine (SAM), etc.), reducing agents (such asdithiothreitol (DTT), beta-mercaptoethanol, etch), salts (e.g., toadjust ionic strength), stabilizing agents (such as serum albumin (e.g.,BSA), size-based exclusion polymers (such as polyethylene glycol (PEG)),and/or the like.

Catalysts can include any material that can increase the rate of achemical reaction (particularly a reaction that modifies nucleic acids)without being consumed or produced by the reaction. Exemplary catalystsfor nucleic acid modification are proteins (enzymes). Any suitableenzymes can be included in reagents. Exemplary enzymes can add singlenucleotides or polynucleotides covalently to a nucleic acid, generallybased on the sequence of a partner strand based-paired with the nucleicacid (i.e., templated addition). Single-nucleotide addition enzymes,which add individual nucleotides successively, generally includepolymerases, such as DNA polymerases (e.g., DNA Polymerase I (orfragments/derivatives thereof (such as the Klenow fragment)),thermostable DNA polymerases (such as Taq Polymerase, Vent Polymerase,Pfu Polymerase, etc.), and/or the like), RNA polymerases (such asphage-derived polymerases (such as SP6, T7, or T3 RNA polymerase),reverse transcriptases that add deoxyribonucleotides based on an RNAtemplate, and/or the like. Polynucleotide addition enzymes, which addtwo or more nucleotides at the same time to a nucleic acid, can includeligases (such as T4 DNA ligase, Taq DNA Ligase, E. coli DNA Ligase,etc.). Other exemplary enzymes can include nucleases (cleavage enzymes),such as restriction enzymes, ribonucleases or deoxyribonucleases (e.g.,single- or double-strand specific enzymes), and/or the like. Otherexemplary catalysts can include polynucleotides (e.g., RNA), syntheticpolymers, transition metal complexes, reactive surfaces, etc.

The reagents can include one or more mono- or polynucleotides forcontact with nanowire assemblies. Mononucleotide reagents can include,for example, nucleoside triphosphates, including deoxyribonucleosidetriphosphates (dNTPs) (e.g., dATP, dCTP, dGTP, dTTP, etc.),ribonucleoside triphosphates (NTPs) (e.g., ATP, CTP, GTP, UTP, etc.),and/or mixtures thereof, among others. Polynucleotide reagents caninclude, for example, nucleic acid dimers, trimers, tetramers, etc. Thepolynucleotide reagents can be configured to be partially or completelycomplementary to a region of the analyte (or to a region being testedfor its presence or absence in the analyte). In some examples, thepolynucleotide reagents can include a 5′-phosphate for ligation to a3′-hydroxyl of a probe.

VI. Controllers

The systems of the present teachings can include at least onecontroller. The controller can interface with (control, coordinate,and/or record) various portions of the systems. For example, thecontroller can interface with operation of the reagent delivery systemand/or the detector, among others.

The controller can interface with any suitable aspect of the reagentdelivery system. In some examples, the controller can control operationof the pump(s), such as determining when the pump is operated, the rateof pump operation, selection of a subset of pumps that are operated,etc. Alternatively, or in addition, the controller can control operationof valves, to determine, for example, which reagent(s) are selected foraddition to a reaction compartment, in what order, at what rate, and/orfor how long, among others. The controller also can receive inputs froma user for user preferences related to operation of the pumps and/orvalves, and/or can store and/or report data related to aspects ofreagent delivery.

The controller can interface with any suitable aspects of thedetector(s). In some examples, the controller can control operation ofthe detector(s), such as determining when the detector is operatedand/or which nanowire assembly is electrically coupled to the detectorat a given time and/or in what order, the size and timing of a back gatevoltage applied to a substrate supporting a nanowire assembly (orassemblies), a voltage and/or current applied to the nanowire assembly,and/or the like. The controller also can receive inputs from a user foruser preferences related to operation of the detector(s), and/or cancollect, store, and/or report data related to aspects of detection.

VII. Examples

The following examples describe selected aspects and embodiments ofsystems for nanowire-based analysis of nucleic acids. These examples areincluded for illustration and are not intended to limit or define theentire scope of the present teachings.

Example 1 Nanowire Arrays

This example describes an exemplary array of nanowire assemblies; seeFIG. 3.

Nanowire array 80 can include a plurality of nanowire assemblies 82supported by a substrate 84. The nanowire assemblies can be disposed ina linear array on the substrate, as shown here, and/or in a two- (orthree-) dimensional array.

Each nanowire assembly can include a nanowire 86 extending betweenspaced electrodes 88. The nanowire can be electrically coupled to theelectrodes, so that current can pass between the electrodes through thenanowire. Exemplary electrodes can be formed of an electricallyconductive material, generally a conductive metal(s) or metal alloy. Inexemplary embodiments, the electrodes can be formed of gold. Electrodescan be formed on the substrate before or after placement of thenanowires. In exemplary embodiments, the electrodes can be formed byphotolithography and/or ion beam lithography.

The substrate can have any suitable composition and structure. In someembodiments, the substrate can be generally planar, and can include anelectrical insulator layer (such as silicon dioxide) disposed over asemiconductor layer (such as silicon). The semiconductor layer can beused to apply a back gate voltage when the nanowire assembly functionsas a field effect transistor (FET), with the electrodes acting as sourceand drain.

Each nanowire assembly can include nucleic acids 92 coupled to ananowire. The nucleic acids can include a probe 94 (such as a primer)and an analyte 96 (such as a template) base-paired with the probe. Theassemblies (or subsets of two or more of the assemblies) can include thesame probe or can include different probes, as shown in the presentillustration. The assemblies (or subsets of two or more of theassemblies) can include the same analyte (or analyte region), as shownin the present illustration, or different analytes.

Example 2 Coupling Nucleic Acids to Nanowires

This example describes exemplary approaches for coupling nucleic acidsto nanowires; see FIGS. 4 and 5.

FIG. 4 shows an exemplary array 100 of nanowire assemblies 102 preparedto receive template 104, shown by the arrow at 106. Each nanowireassembly can include a nanowire 108 and a different probe 110 coupled tothe nanowire. The probes can be coupled to the nanowires before or afterthey are disposed in the array. Templates 104 can be placed in contactwith the array and allowed to hybridize with the probes and/or can becoupled directly to the nanowires. Accordingly, the probes can beconfigured to select complementary templates from a nucleic acid mixtureof templates and nontemplate species. After hybridization, unpairedtemplates (and nontemplate species) can be removed.

FIG. 5 shows an exemplary array 120 of nanowire assemblies 122 preparedto receive primers 124, shown by the arrow at 126. Each nanowireassembly can include a nanowire 128 and a different (or the same)template 130 coupled to the nanowire. The templates can be coupled tothe nanowires before or after the nanowires are disposed in the array.Primers 124 can be placed in contact with the array and allowed tohybridize with the templates and/or can couple directly to thenanowires. Accordingly, the templates can be configured to selectcomplementary primers from a primer mixture. After hybridization,unpaired (e.g., excess) primer molecules can be removed.

Example 3 System for Nanowire-Based Sequencing

This example describes an exemplary system, including apparatus, method,and data, for nanowire-based sequencing; see FIGS. 6-8.

A system 140 for nanowire based sequencing can include a reagentdelivery system 142 in fluid communication with a reaction compartment144 holding an array 146 of nanowire assemblies 148. The system also caninclude a detector 150 electrically coupled (or couplable) to the arrayof nanowire assemblies, and a controller 152 in communication with thedetector and the reagent delivery system.

Reagent delivery system 142 is configured to move fluid through reactioncompartment 144 for contact with array 146. The reagent delivery systemthus can include a plurality of fluid reagents 154 for covalentmodification of nucleic acids, such as four different nucleosidetriphosphates (dNTPs) 156, 158, 160, and 162, each disposed in anaqueous solution with DNA polymerase molecules 164. The fluid reagentsalso can include a wash reagent 166, which can be used, for example, towash dNTP reagents out of the reaction compartment after they havecontacted the nanowire assemblies. Movement of the fluid reagents can bedriven by a pump 168 disposed, for example, downstream of the fluidreagents. The pump can drive each fluid reagent to (and past) thereaction compartment. Fluid reagents can be selected according toselective opening and closing of valves 170 disposed between reagentreservoirs 172 and the reaction compartment. The reagent delivery systemcan define a plurality of compartments for holding fluid, including thereagent reservoirs, the reaction compartment, a waste reservoir 174disposed downstream of the reaction compartment, and channels 176extending between and/or defining these structures. In some examples,the reagent delivery system can be disposed on a planar substrate, withmicrofluidic (or larger) passages for holding and carrying fluid formedon and/or above the planar substrate by a fluidics layer abutted to thesubstrate.

Nanowire array 146 can include nanowire assemblies each coupled tonucleic acids corresponding to a template 178 base-paired with a primer180. In operation, the nucleotide addition reagents 156-162 can beindividually dispensed to the reaction compartment. Before, during,and/or after each addition, the detector can measure the conductance ofeach nanowire assembly, allowing a determination of whether or not eachnucleotide was attached to the primer based on the sequence of thetemplate, and, if added, how many subunits were attached.

FIG. 7 shows an exemplary method 200 of sequencing nucleic acidtemplates, which can be performed with the sequencing system shown inFIG. 6. The method can include a step of providing a nanowire assembly,shown at 202. The nanowire assembly can include a nanowire, a template,and a primer for the template. The nanowire assembly can be part of anarray of assemblies. The method can include a step of contacting thenanowire assembly with a nucleotide addition reagent, shown at 204. Thenucleotide addition reagent can include, for example, a polymerase andnucleoside triphosphate. The method can include a step of measuring,shown at 206. Measuring can detect an electrical characteristic of eachnanowire assembly before, during, and/or after the step of contacting todetermine whether or not one or more nucleotides were attached(covalently) to each nanowire assembly. The steps of contacting andmeasuring can be repeated for the other nucleotide reagents (or only asubset thereof), shown at 208. In some examples, the steps can berepeated until each of the four different nucleotide reagents (or asuitable subset thereof, such as two different nucleotide reagentscorresponding to two known single nucleotide polymorphisms) haveseparately contacted the nanowire assemblies. If only a singlenucleotide position of the template, immediately adjacent the end of theprimer, is of interest, the method can be terminated, shown at 210.However, if the controller determines that the template can and/orshould be sequenced more, to obtain sequence information aboutadditional nucleotide positions in the template, the steps ofcontacting, measuring, and repeating can be performed again, shown at212, during one more additional cycles.

FIG. 8 shows exemplary data that may be obtained using method 200 ofFIG. 7. Each of the four dNTPs (including polymerase) are dispensedstepwise, shown at 220, to a pair of nanowire assemblies 222. Graph 224plots each nucleotide reagent dispensed, shown at 226, against theconductance measured after each addition, shown at 228. An increase inconductance during and after contacting with a particular nucleotidesignifies a partner nucleotide, complementary to the particularnucleotide, at the corresponding position in the template strand. Thesize of the increase signifies how many nucleotide subunits wereattached, with the increase being generally proportional to the numberof nucleotide subunits attached to each primer molecule of a nanowireassembly. Accordingly, the upper plot, shown at 230, indicates covalentaddition of AGGTTCAA, and the lower plot, shown at 232, indicatescovalent addition of GAGTCCA (and thus the presence of complementarysequences at corresponding regions of the template).

Example 4 Nanowire-Based Analysis Utilizing Ligation

This example describes nucleic acid analysis using ligation of nucleicacids associated with nanowires; see FIG. 9.

Humans have substantially identical genomes. Accordingly, positions ofsequence variation within the population (e.g., single-nucleotidepolymorphisms) can be utilized to identify individuals or lineages(e.g., for forensic purposes), to diagnose genetic diseases orconditions, and/or to predict responses to treatment regimens (e.g., tofacilitate selection of drugs), among others. Ligation-based analysiscan be suitable for sequencing individual positions of sequencevariation in the population. In particular, ligation-based analysis canutilize the largely known sequence of a nucleic acid analyte to generateprobes that allow sequencing of nucleotide positions of known variationamong individuals of a population.

FIG. 9 shows a flowchart 250 for a method of nanowire-based sequencingusing ligation, and exemplary data that may be obtained using themethod.

Nanowire assemblies 252, 254 can be provided. The assemblies can includea nanowire 256 and a different probe 258, 260 coupled to each nanowire.The probes can include a region of nucleotide sequence variation in thehuman population. In particular, the probes (or a region thereof) candiffer by only one (or a few) nucleotides, with each probe correspondingto a different sequence version of a polymorphism in the population.Furthermore, the sequence differences between the probes can be disposedat or near the end of the probes, so that any nonpairing of the probewith a template will be pronounced at the end of probe.

A template 262 having a polymorphic nucleotide 264 (in the population)can contact, shown at 266, the nanowire assemblies. The template canbase pair with each of the probes to form nucleic acid duplexes 268,270. However, probe 258 can be partially unpaired in duplex 268, shownat 272, because this probe does not form a base pair with thepolymorphic nucleotide. Probe 260 is fully paired with the template induplex 2707 shown at 274, because this probe does form a base pair withthe polymorphic nucleotide.

An extension nucleic acid 276 and a ligase enzyme can contact, shown at278, S the nanowire assemblies. The extension nucleic acid can be a(5′-phosphorylated) oligonucleotide configured to pair with the templatein a position adjoining each probe. Accordingly, apposed ends of theextension nucleic acid and the probe can be joined by the ligase enzyme,shown at 280, to produce a ligation product 282 that is an extendedversion of probe 260 in assembly 283. However, ends of the extensionnucleic acid and probe 258 are not substantially apposed for efficientligation in assembly 284.

Nucleic acid duplexes can be disrupted, shown at 285. This disruptioncan leave original probe 258 and ligation product 282 coupled to theirrespective nanowires, while releasing the template from both nanowires,and the (unligated) extension nucleic acid from only the upper nanowire.

Conduction of the nanowires can be tested before and after the series ofoperations, shown at 286 and 288, respectively. Exemplary data that maybe obtained is plotted in a graph 290. Probe 258 was not lengthened byligation, so conductance of its associated nanowire is not changed,shown at 292. Probe 260 was extended by ligation, so conductance of itsassociated nanowire is increased, shown at 294.

The method presented above can be modified in various ways. For example,a single probe can be used sequentially with different extensions. Inparticular, the probe can hybridize with the template adjacent thepolymorphic nucleotide, and then different potential extensionsubstrates, which hybridize to different versions of the polymorphicnucleotide can be added separately, and tested for their ability to beligated to the probe.

Further illustrative discussion of ligation conditions and other aspectsof ligation are described, for example, in U.S. Pat. No. 6,511,810 (suchas at column 17, line 58. to column 18, line 47), which is incorporatedherein by reference

Example 5 Nanowire-Based Analysis with Cleavage

This example describes an exemplary nanowire-based system usingselective cleavage of nucleic acids with a nuclease; see FIG. 10.

Nanowire assemblies 300, 302 including distinct nucleic acid duplexes304, 306 can be distinguished by differential cleavage of the duplexes,shown at 308, 310, using a nuclease. The nuclease can selectively cutduplexes according to primary sequence, base-pair mismatches, unpairedends, presence of a duplex, absence of a duplex, and/or the like.Differential cleavage of probes 312, 314 can be detected by measuringconductance of the nanowire assemblies with or without a separate duplexdisruption step. In some examples, appropriate selection of probes canallow cleavage to be detected as a cleavage-induced destabilization ofthe duplex, as shown in the present illustration.

Example 6 Exemplary Coupling of Nucleic Acids to Nanowires

This example describes an exemplary approach for coupling nucleic acidsto nanowires at two or more sites along each nucleic acid; see FIG. 11.

Each nucleic acid (i.e., an analyte and/or a probe) can be coupled to ananowire at one or more positions along the nucleic acid. In someexamples, the nucleic acid can be coupled at two or more spaced sites(i.e., separated by one or more nucleotides of the nucleic acid).Coupling at multiple sites can constrain the nucleic acid to anorientation that is more parallel to the nanowire than coupling at asingle site. Accordingly, the use of multiple coupling sites canposition the nucleic acid closer to the nanowire, with less variation inspacing from the nanowire for different regions of the nucleic acid.Placing the nucleic acid closer to the nanowire, and in a moreconstrained configuration, can increase the sensitivity with whichchanges in an electrical characteristic can be measured, and also canreduce the variation in the changes measured with respect to differentregions of the nucleic acid. As a result, the use of multiple couplingsites per nucleic acid molecule can allow a number of advantages oversingle-site coupling, such as (1) more sequence information (i.e.,longer reads) for each analyte, (2) sequence analysis with fewer analytemolecules per nanowire, and/or (3) more consistent sequence analysis ofanalytes, among others.

FIG. 11 shows an exemplary nanowire assembly 350 during coupling ofmolecules of a nucleic acid 352 (an analyte and/or a probe) to ananowire 354 of the assembly at two or more sites (e.g., sites 356, 358)along the nucleic acid. Each coupling site can be formed by a specificbinding pair 360 (see Section III above). For example, nanowire 354 canbe connected to a first binding member 362, and nucleic acid 352 can beconnected to a second binding member 364 that binds specifically to thefirst binding member. Each molecule of the nucleic acid can be connectedto (or integrally include) two or more moieties of the second bindingmember, to provide two or more binding sites along the nucleic acid. Themoieties can be spaced from one another, for example, disposed generallytoward opposing ends of the nucleic acid, to facilitate tethering two ormore distinct regions of the nucleic acid to the nanowire. Exemplaryfirst and second binding members can include, respectively, (1)streptavidin and biotin, (2) biotin and streptavidin, and/or (3)complementary nucleic acids, among others. If base-pairing is used tocouple the nucleic acid to the nanowire at two or more spaced sites,spaced regions of the nucleic acid itself can be used for base-pairinginteraction. A base-paired partner disposed at one or more of the spacedregions (and connected more directly to the nanowire) also can serve asa probe/primer, or a distinct probe/primer can be hybridized to thenucleic acid.

The disclosure set forth above may encompass multiple distinctinventions with independent utility. Although each of these inventionshas been disclosed in its preferred form(s), the specific embodimentsthereof as disclosed and illustrated herein are not to be considered ina limiting sense, because numerous variations are possible. The subjectmatter of the inventions includes all novel and nonobvious combinationsand subcombinations of the various elements, features, functions, and/orproperties disclosed herein. The following claims particularly point outcertain combinations and subcombinations regarded as novel andnonobvious. Inventions embodied in other combinations andsubcombinations of features, functions, elements, and/or properties maybe claimed in applications claiming priority from this or a relatedapplication. Such claims, whether directed to a different invention orto the same invention, and whether broader, narrower, equal, ordifferent in scope to the original claims, also are regarded as includedwithin the subject matter of the inventions of the present disclosure.

1. A method of analyzing a nucleic acid, comprising: coupling asemiconductor nanowire to a nucleic acid analyte to form a nanowireassembly, the nanowire coupled to first and second electrodes; inducinga current through the nanowire assembly; contacting the nanowireassembly with at least one reagent, wherein the reagent covalentlymodifies the nucleic acid analyte based on the nucleic acid's structure;measuring an electrical characteristic of the nanowire assembly todetermine whether the at least one reagent produced a covalentmodification of the nanowire assembly; and providing structuralinformation about the nucleic acid analyte.
 2. The method of claim 1,further comprising contacting a plurality of nanowire assemblies with afluid that includes the at least one reagent.
 3. The method of claim 2,wherein the nanowire assemblies are coupled to different nucleic acidanalytes, and wherein the step of measuring provides structuralinformation about each of the different nucleic acid analytes.
 4. Themethod of claim 1, wherein the step of measuring includes a step ofmeasuring substantially no change in the electrical characteristic,thereby indicating that the nucleic acid analyte lacks structure capableof directing covalent modification by the at least one reagent.
 5. Themethod of claim 1, wherein the step of coupling comprises coupling thesemiconductor nanowire to a nucleic acid probe and selectively pairingthe nucleic acid probe to the nucleic acid analyte.
 6. The method ofclaim 5, wherein the nucleic acid probe is configured as a primer ofnucleic acid synthesis with the nucleic acid analyte as a template, andwherein the step of contacting includes a step of contacting the primerand the template with a polymerase and a nucleotide substrate for thepolymerase.
 7. The method of claim 6, wherein the step of measuringdetermines an amount of change in the electrical characteristic, if any,the method further comprising a step of correlating the amount to anumber of nucleotide subunits linked to the primer by the step ofcontacting.
 8. The method of claim 6, wherein the steps of contactingand measuring are repeated individually for each of four differentnucleotide substrates corresponding to the nucleotides guanosine,adenosine, thymidine, and cytidine.
 9. The method of claim 8, whereinthe steps of contacting and measuring are repeated over two or morecycles of contacting individually with the four different nucleotidesubstrates.
 10. The method of claim 1, wherein the step of contactingincludes a step of contacting with a reagent having ligation activity,and wherein the step of measuring includes a step of determining whetheror not nucleic acid ligation occurred.
 11. The method of claim 1,wherein the step of contacting includes a step of contacting with areagent for cleavage of nucleic acids, and wherein the step of measuringincludes a step of determining whether or not nucleic acid cleavageoccurred.
 12. The method of claim 1, further comprising a step ofadjusting a stringency for nucleic acid hybridization.
 13. The method ofclaim 12, wherein the step of adjusting a stringency is performed byadjusting at least one of a temperature, an electric field, and a fluidcomposition associated with the nanowire assembly.
 14. The method ofclaim 1, wherein the step of contacting is performed with a nanowirecoupled to a nucleic acid at two or more spaced positions along thenucleic acid.
 15. The method of claim 1, wherein the nanowire functionsas a field effect transistor.
 16. The method of claim 1, furthercomprising electrically coupling the nanowire to the first and secondelectrodes.
 17. The method of claim 1, wherein the covalent modificationalters the current passing through the nanowire assembly.
 18. The methodof claim 1, further comprising applying a voltage to the nanowireassembly.
 19. The method of claim 18, further comprising gating thevoltage to the nanowire assembly.
 20. The method of claim 1, furthercomprising applying a back-gate voltage to the nanowire assembly. 21.The method of claim 1, wherein the step of measuring comprises measuringthe conductance of the nanowire assembly.
 22. The method of claim 1,wherein the step of coupling comprises contacting the nanowire assemblywith a nucleic acid primer that pairs selectively with the nucleic acidanalyte to couple the nucleic acid primer to the nanowire.
 23. A methodof analyzing a nucleic acid, comprising: coupling a nucleic acid analyteto a semiconductor nanowire to form a nanowire assembly, the nanowirecoupled to first and second electrodes; contacting the nanowire assemblywith at least one reagent, the at least one reagent modifying thenucleic acid analyte based on a structure of the nucleic acid analyte;and determining whether the at least one reagent modified the nucleicacid analyte based on a change in an electrical characteristic of thenanowire assembly.
 24. The method of claim 23, wherein the at least onereagent includes a nucleic acid monomer.
 25. A method of analyzing anucleic acid, comprising: coupling a nucleic acid analyte to asemiconductor nanowire, the nanowire coupled to first and secondelectrodes; contacting the nucleic acid analyte with at least onenucleic acid monomer, the at least one nucleic acid monomer covalentlymodifying the nucleic acid analyte based on a structure of the nucleicacid analyte; determining whether the at least one nucleic acid monomercovalently modified the nucleic acid analyte based on a change in anelectrical characteristic of the nanowire; and determining a portion ofthe structure of the nucleic acid analyte based on determining whetherthe at least one nucleic acid monomer covalently modified the nucleicacid analyte.